Bacillus anthracis spores are an appealing biological weapon, which can be distributed through many different means to cause bioterrorism, such as the intentional contamination of the U.S. mail after the September 11 terrorist attacks. Anthrax vaccine adsorbed (AVA), the only human anthrax vaccine in the U.S., was licensed for the prevention of cutaneous anthrax and requires initial multiple injections with subsequent annual boosters. Thus, there is a critical need to develop an efficacious and easily administrable vaccine to protect against anthrax infection, especially the inhalation anthrax, the most lethal form of anthrax. New anthrax vaccine development has been focused on the production of a vaccine based on purified protective antigen (PA) protein of the anthrax toxin. However, it was shown that, besides the PA, other antigens such as the lethal factor (LF), a protein that binds to PA to form anthrax lethal toxin, and the poly-gamma-D-glutamic acid (PGA) capsule surrounding the vegetative anthrax bacilli, should also be included in an anthrax vaccine for more effective protection. A nasal multi- antigen anthrax vaccine will be advantageous not only because it will be ease to administer, but also because it is expected to induce immune responses against all antigens in both the systemic compartment and the mucosal tissues of the respiratory tract, through which inhaled anthrax spores enter hosts. Data from recent studies have indicated the importance of the anti-PA mucosal responses in preventing against inhalational anthrax challenges in animals. We propose to develop an efficacious nasal anthrax vaccine that can induce specific systemic and mucosal immune responses against both the anthrax toxins and the vegetative bacilli by incorporating a 63 kDa form of PA, an inactivated LF mutant, and PGA as antigens into a lipid-based particulate vaccine carrier we have developed. We hypothesize that that this tri-antigen nasal vaccine will be more effective in preventing against an inhalational anthrax infection than the systemic injection of a vaccine based on PA alone. Our specific aims are to define the immune responses induced by this tri-antigen nasal anthrax vaccine in mice (aim 1), to evaluate the anti-toxin and anti-bacillus activities induced by it (aim 2), and to validate the efficacy of this vaccine in preventing against a pulmonary anthrax spore challenge in mice (aim 3). The completion of this project is expected provide a sound scientific foundation for further developing this vaccine into an efficacious and easily administrable nasal anthrax vaccine for humans. The development of a prototype nasally administrable multi-antigen anthrax vaccine that targets both anthrax toxins and bacilli and the validation of its efficacy in a murine model will provide a sound foundation for the development of a new generation human anthrax vaccine, which is urgently needed for effective mass immunization in case of a covert anthrax outbreak. The same vaccine or its modified version can also be used in agricultural industries to vaccinate cattle. [unreadable] [unreadable] [unreadable]